1. Field of the Invention
The present invention relates to novel complexes of major histocompability complex (MHC) molecules and methods of expressing and use of such complexes. For example, in one aspect, the invention relates to MHC class II molecules that include a modified class II .beta.2 chain. In another aspect, the invention relates to MHC class I and class II complexes that include a covalently linked immunoglobin constant region. In still other aspects, the invention relates to polyspecific MHC complexes, as well as methods of expressing and purifying MHC complexes. The MHC complexes of the invention are useful for a variety of applications including screening peptides for the capacity to modulate T-cell activity in vitro and in vivo.
2. Background
Antigen-specific T-cell responses are invoked by antigenic peptides. The peptides generally bind to the binding groove of MHCs as part of an immune system mechanism for identifying and responding to foreign antigens. The bound antigenic peptides interact with T-cell receptors and modulate an immune response. The antigenic peptides are bound by non-covalent means to particular "binding pockets" comprised of polymorphic amino acid residues.
Naturally-occurring MHC class II molecules are heterodimeric glycoproteins consisting of .alpha. and .beta. chains. The .alpha.1 and .beta.1 domains of these molecules fold together to form a peptide binding grove. Antigenic peptides bind the MHC molecule through interaction between anchor amino acids on the peptide and the .alpha.1 and .beta.1 domains. Crystallographic analysis of human class II HLA-DR1 complex bound to an influenza virus peptide indicates that the N- and C-terminal ends of the bound peptide extend out of the binding groove such that the C-terminus of the peptide is proximal to the N-terminus of the .beta. chain. See e.g., J. Brown et al., Nature, 364:33 (1993); L. Stern et al., Nature, 368:215 (1994)). MHC class I and class II molecules have different domain organizations. See e.g., A. Rudensky et al., Nature, 353:622 (1991). See also U.S. Pat. Nos. 5,284,935; 5,260,422; 5,194,425; 5,130,297; WO 92/18150; WO 93/10220; and WO96/04314 for discussions of MHC molecules.
Particularly, J. Brown, et al. supra have reported that the MHC class II .beta.2 chain performs a critical role in the proper folding of MHC class II complexes.
The .alpha. and .beta. chain transmembrane domains play an important role in the assembly and/or intracellular transport of MHC molecules. For example, amino acid changes in the TM domains can result in defective MHC molecules. The MHC .alpha. and .beta. chain transmembrane and cytoplasmic domains have been disclosed. See P. Cosson et al., Science, 258:659 (1992); W. Wade et al., Immunology, 32:433 (1995); H. Kozono et al., Nature, 369:151 (1994) and J. Brown et al., supra.
MHC molecules complexed with antigenic peptides can induce selective immunosuppression by several different mechanisms. See e.g., J. Guery et al., Critical Reviews in Immunology, 13(3/4):195 (1993)).
More specifically, it has been reported that peptide-MHC complexes on the surface of antigen presenting cells (APCs) will only induce clonal expansion of a T-cell line specific for the MHC bound peptide if the antigen presenting cells also deliver co-stimulatory signals. One proposed approach takes advantage of this requirement for T-cell activation and reports inhibition of T-cell development by interaction with the antigenic peptide bound to the MHC molecule in the absence of co-stimulatory signals. See M. Nicolle et al., J. Clin. Invest., 93:1361-1369 (1994); and S. Sharma et al., Proc. Natl. Acad. Sci. USA, 88:11465-11469 (1991).
Another proposed approach includes inhibiting T-cell development with MHC molecules that contain a bound peptide. The bound peptide can be an antagonist or partial agonist to a T-cell receptor (TCR). See B. Evavold et al., Immunology Today, 14(12):602-609 (1993).
Modifications of TCR-bound antigenic peptides have been attempted to examine residues responsible for specific T-cell responses. Determination of such "activating" amino acids of the antigenic peptides could provide insight into those amino acid sequences which can potentially play roles as TCR agonists or antagonists. See Evavold, B. et al., supra.
It also has been speculated that new vaccines might be developed based on determination of the nature of various antigenic peptides bound to MHC molecules. See R. Chicz et al., Immunology Today, 15(4):155-160 (1994).
Previous studies have shown that MHC class II heterodimeric molecules can bind exogenous peptide. However, the MHC class II chains often dissociate. In a dispersed state, the MHC class II chains may not be suitable for binding presenting peptide. See Stern, L. J. and D. C. Wiley, Cell 68: 465 (1992); Scheirle, A. B. et al., J. Immunol. 149: 1994 (1992); Kozano H. et al., Nature 369:151 (1994).
There have been several attempts to obtain fully soluble and functional MHC complexes. For example, in one approach, MHC complexes have been isolated from cells using biochemical techniques that include exposure to harsh agents such as proteolytic enzymes, salts, and/or detergents. These agents must often be removed by dialysis or binding reactions. See e.g., J. M Turner et al. J. Biol. Chem. 252: 7555 (1977); T. A. Springer et al. PNAS (USA) 73: 2481(1976).
However, these methods are often not optimal for isolating fully soluble and functional MHC complexes in significant quantities.
Highly useful MHC class I and class II complexes capable of modulating the activity of T-cells and methods of making the complexes have been disclosed in a published PCT Application No. WO 96/04314, filed Jul. 31, 1995. The MHC complexes disclosed generally bind a specific peptide ligand.